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Volume 223, Issue 2 p. 351-357
Free Access

Purification of the AMP-activated protein kinase on ATP-γ-Sepharose and analysis of its subunit structure

Stephen P. DAVIES

Stephen P. DAVIES

Department of Biochemistry, The University, Dundee, Scotland

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Simon A. HAWLEY

Simon A. HAWLEY

Department of Biochemistry, The University, Dundee, Scotland

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Angela WOODS

Angela WOODS

MRC Molecular Medicine Group, Royal Postgraduate Medical School, Hammersmith Hospital, London, England

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David CARLING

David CARLING

MRC Molecular Medicine Group, Royal Postgraduate Medical School, Hammersmith Hospital, London, England

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Timothy A. J. HAYSTEAD

Timothy A. J. HAYSTEAD

Department of Pharmacology, University of Virginia, Charlottesville VA, USA

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D. Grahame HARDIE

Corresponding Author

D. Grahame HARDIE

Department of Biochemistry, The University, Dundee, Scotland

Correspondence to D. G. Hardie, Department of Biochemistry, The University, Dundee, Scotland DD1 4HNSearch for more papers by this author
First published: July 1994
Citations: 119

Abstract

The AMP-activated protein kinase has been purified by affinity chromatography on ATP-γ-Sepharose. A proportion of the activity can be eluted using AMP, while the remainder is eluted using ATP. The AMP eluate contains three polypeptides of 63, 38 and 35 kDa (p63, p38 and p35) in a molar ratio (by Coomassie blue binding) close to 1:1:1. p63 was previously identified as the AMP-binding catalytic subunit [Carling, D., Clarke, P. R., Zammit, V. A. & Hardie, D. G. (1989) Eur. J. Biochem. 186, 129–136]. All three polypeptides exactly comigrate both on native gel electrophoresis and on gel filtration, suggesting that p38 and p35 are additional subunits. Estimation of Stokes radius (5.4–5.8 nm) by gel filtration, and sedimentation coefficient (7.9–8.4 S) by glycerol gradient centrifugation, suggest that the kinase has an asymmetric structure with a native molecular mass for the complex of 190 ± 10 kDa. Thus the native enzyme appears to be a heterotrimer with a p63/p38/p35 (1:1:1) structure. Despite the fact that the ATP eluate has a higher specific activity than the AMP eluate (3.5 ± 0.2 vs 2.3 ± 0.2 μmol · min−1· mg−1), it appears to be less pure, containing p63, p38 and p35 plus other polypeptides. Experiments examining the effects of protein phosphatase-2A and kinase kinase, and analysis by Western blotting with anti-p63 antibody, suggests that the AMP eluate is entirely in the low-activity dephosphorylated form, while the ATP eluate is a mixture of that form and the high-activity phosphorylated form. As well as establishing the subunit structure of the AMP-activated protein kinase, these results suggest that the kinase can bind to ATP-γ-Sepharose through either the allosteric (AMP/ATP) site or the catalytic (ATP) site, and that phosphorylation by the kinase kinase increases the affinity for the latter site.

Abbreviations

  • HMG-CoA
  • 3-hydroxy-3-methylglutaryl-coenzyme A
  • AMPK
  • AMP-activated protein kinase
  • PP2A
  • protein phosphatase-2A
  • PP2C
  • protein phosphatase-2C
  • Enzymes

  •  
  • 3-Hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34)
  •  
  • 3-hydroxy-3-methylglutaryl-CoA reductase kinase, AMP-activated protein kinase (EC 2.7.1.109)
  •  
  • acetyl-CoA carboxylase (EC 6.4.1.2)
  •  
  • acetyl-CoA carboxylase kinase (EC 2.7.1.128)
  •  
  • hormone-sensitive lipase (EC 3.1.1.3)
  •  
  • sterol esterase (EC 3.1.1.13)
  •  
  • protein phosphatases −2A and −2C (EC 3.1.3.16)